Fluorogen-activating proteins: beyond classical fluorescent proteins

Xu, Shengnan; Hu, Hai-Yu

In 2014, the Nobel Prize in Chemistry recognized super resolution imaging, a breakthrough in optical microscopy that enables researchers to visualize and investigate biological processes at the individual molecule level inside living cells. Fluorogen-based reporters were furthermore shown to open great prospects for super-resolution microscopy and single molecule tracking. Highly photostable far-red MG-based FAPs fluoromodules were reported to be well suitable for live cell imaging with stimulated emission depletion (STED) nanoscopy in mammalian cells and bacteria 23, 50 . The ability to control the labeling density of the FAPs by adjusting the fluorogen concentration in the milieu was used to obtain sparse labeling distribution of densely genetically tagged proteins for single molecule localization imaging in living cells, further demonstrating the interest of such probes for super-resolution microscopy 22 . This system is advantageous over traditional approaches 51, 52 , because it does not require special imaging buffers or photo-activation or photoswitching with a second laser line. The ability to label a subset of proteins independently of the expression level also enabled to track individual receptors in living cells 53 , confirming the usefulness of FAPs for single molecule studies.